Journal of Biological Chemistry

doi: 10.1016/s0021-9258(20)45783-7pmid: N/A

doi: 10.1016/s0021-9258(20)45718-7pmid: N/A

Malhi, Harmeet;Kropp, Erin M.;Clavo, Vinna F.;Kobrossi, Christina R.;Han, JaeSeok;Mauer, Amy S.;Yong, Jing;Kaufman, Randal J.

doi: 10.1074/jbc.m112.442954pmid: 23720735

<p>Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. <i>Chop</i> wild type (<i>Chop</i>^+/+) and knock-out (<i>Chop</i>^−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, <i>Chop</i>^−/− mice developed greater liver injury, inflammation, and fibrosis than <i>Chop</i>^+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from <i>Chop</i>^+/+ and <i>Chop</i>^−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated <i>Chop</i>^+/+ and <i>Chop</i>^−/− macrophages, <i>Chop</i>^−/− macrophages were resistant to its lipotoxicity. <i>Chop</i>^−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between <i>Chop</i>^−/− and <i>Chop</i>^+/+ mice demonstrated that <i>Chop</i> in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that <i>Chop</i> deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.</p><p>Background: We hypothesized that C/EBP homologous protein mediates hepatocyte apoptosis in nonalcoholic steatohepatitis.</p><p>Results: Paradoxically, <i>Chop</i> deletion protects from steatohepatitis by inducing apoptosis in activated macrophages.</p><p>Conclusion: CHOP-dependent macrophage apoptosis in NASH highlights the cell type-specific complexity of the ER stress response.</p><p>Significance: Therapeutic manipulation of mediators of ER stress response may have opposite effects in different cell populations; therefore, such studies should be interpreted cautiously.</p>

Vannier, Corinne;Mock, Kerstin;Brabletz, Thomas;Driever, Wolfgang

doi: 10.1074/jbc.m113.467787pmid: 23667256

<p>The ZEB1 transcription factor is best known as an inducer of epithelial-mesenchymal transitions (EMT) in cancer metastasis, acting through transcriptional repression of <i>CDH1</i> (encoding E-cadherin) and the EMT-suppressing microRNA-200s (miR-200s). Here we analyze roles of the ZEB1 zebrafish orthologs, Zeb1a and Zeb1b, and of miR-200s in control of cell adhesion and morphogenesis during gastrulation and segmentation stages. Loss and gain of function analyses revealed that Zeb1 represses <i>cdh1</i> expression to fine-tune adhesiveness of migrating deep blastodermal cells. Furthermore, Zeb1 acts as a repressor of <i>epcam</i> in the deep cells of the blastoderm and may contribute to control of epithelial integrity of enveloping layer cells, the outermost cells of the blastoderm. We found a similar ZEB1-dependent repression of <i>EPCAM</i> expression in human pancreatic and breast cancer cell lines, mediated through direct binding of ZEB1 to the <i>EPCAM</i> promoter. Thus, Zeb1 proteins employ several evolutionary conserved mechanisms to regulate cell-cell adhesion during development and cancer.</p><p>Background: Regulation of cell adhesion is important for embryonic development and to prevent cancer metastasis.</p><p>Results: Zeb1 controls cell adhesion in zebrafish embryos and human cancer cell lines through transcriptional repression of E-cadherin, Epcam, and miR-200s.</p><p>Conclusion: Zeb1 fine-tunes E-cadherin- and Epcam-mediated cell adhesion to control cell behavior during gastrulation.</p><p>Significance: Conserved cell adhesion regulation mechanisms are crucial for understanding development and cancer invasion.</p>

Hurst, Jillian H.;Dohlman, Henrik G.

doi: 10.1074/jbc.m113.475707pmid: 23645675

<p>Ubiquitination is a post-translational modification that tags proteins for proteasomal degradation. In addition, there is a growing appreciation that ubiquitination can influence protein activity and localization. Ste7 is a prototype MAPKK in yeast that participates in both the pheromone signaling and nutrient deprivation/invasive growth pathways. We have shown previously that Ste7 is ubiquitinated upon pheromone stimulation. Here, we show that the Skp1/Cullin/F-box ubiquitin ligase SCF^Cdc4 and the ubiquitin protease Ubp3 regulate Ste7 ubiquitination and signal specificity. Using purified components, we demonstrate that SCF^Cdc4 ubiquitinates Ste7 directly. Using gene deletion mutants, we show that SCF^Cdc4 and Ubp3 have opposing effects on Ste7 ubiquitination. Although SCF^Cdc4 is necessary for proper activation of the pheromone MAPK Fus3, Ubp3 is needed to limit activation of the invasive growth MAPK Kss1. Finally, we show that Fus3 phosphorylates Ubp3 directly and that phosphorylation of Ubp3 is necessary to limit Kss1 activation. These results reveal a feedback loop wherein one MAPK limits the ubiquitination of an upstream MAPKK and thereby prevents spurious activation of a second competing MAPK.</p><p>Background: Ubiquitination is a post-translational modification that regulates protein behavior.</p><p>Results: Pheromone stimulation induces dynamic ubiquitination of the MAPKK Ste7; disruption of this modification leads to altered MAPK signal specificity.</p><p>Conclusion: Dynamic ubiquitination is required to maintain the strength and fidelity of the pheromone response.</p><p>Significance: This study identifies a novel regulatory mechanism in MAPK cascades, a signaling module that is central to human physiology and disease.</p>

doi: 10.1074/jbc.p113.475707pmid: N/A

♦ See referenced article, J. Biol. Chem. 2013, 288, 18660–18671

Gu, Lei;Liu, Cong;Guo, Zhefeng

doi: 10.1074/jbc.m113.457739pmid: 23687299

<p>Oligomerization of the 42-residue peptide Aβ42 plays a key role in the pathogenesis of Alzheimer disease. Despite great academic and medical interest, the structures of these oligomers have not been well characterized. Site-directed spin labeling combined with electron paramagnetic resonance spectroscopy is a powerful approach for studying structurally ill-defined systems, but its application in amyloid oligomer structure study has not been systematically explored. Here we report a comprehensive structural study on a toxic Aβ42 oligomer, called globulomer, using site-directed spin labeling complemented by other techniques. Transmission electron microscopy shows that these oligomers are globular structures with diameters of ∼7–8 nm. Circular dichroism shows primarily β-structures. X-ray powder diffraction suggests a highly ordered intrasheet hydrogen-bonding network and a heterogeneous intersheet packing. Residue-level mobility analysis on spin labels introduced at 14 different positions shows a structured state and a disordered state at all labeling sites. Side chain mobility analysis suggests that structural order increases from N- to C-terminal regions. Intermolecular distance measurements at 14 residue positions suggest that C-terminal residues Gly-29–Val-40 form a tightly packed core with intermolecular distances in a narrow range of 11.5–12.5 Å. These intermolecular distances rule out the existence of fibril-like parallel in-register β-structures and strongly suggest an antiparallel β-sheet arrangement in Aβ42 globulomers.</p><p>Background: Aβ42 oligomers underlie neurotoxicity in Alzheimer disease, but their molecular structures are unknown.</p><p>Results: Electron paramagnetic resonance studies reveal intermolecular distances at 11.5–12.5 Å for C-terminal region of Aβ42.</p><p>Conclusion: Aβ42 oligomers consist of a tightly packed C-terminal region that adopts antiparallel structures.</p><p>Significance: This work provides insights into the structures of Aβ42 oligomers and helps understand their oligomerization mechanism and toxicity.</p>

doi: 10.1074/jbc.p113.457739pmid: N/A

♦ See referenced article, J. Biol. Chem. 2013, 288, 18673–18683

Bhattacharjee, Arnab;Oeemig, Jesper S.;Kolodziejczyk, Robert;Meri, Taru;Kajander, Tommi;Lehtinen, Markus J.;Iwaï, Hideo;Jokiranta, T. Sakari;Goldman, Adrian

doi: 10.1074/jbc.m113.459040pmid: 23658013

<p><i>Borrelia burgdorferi</i> spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of <i>B. burgdorferi</i> is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how <i>B. burgdorferi</i> evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.</p><p>Background: <i>Borrelia burgdorferi</i> OspE protein recruits complement regulator FH onto the bacteria for immune evasion.</p><p>Results: We solved the structure of OspE and the OspE·FH complex by NMR and x-ray crystallography.</p><p>Conclusion: The OspE·FH structure shows how <i>Borrelia</i> evade complement attack by mimicking how host cells protect themselves.</p><p>Significance: This explains how the bacteria survive in the host and facilitates vaccine design against borreliosis.</p>

Arimori, Takao;Kawamoto, Noriko;Shinya, Shoko;Okazaki, Nobuo;Nakazawa, Masami;Miyatake, Kazutaka;Fukamizo, Tamo;Ueda, Mitsuhiro;Tamada, Taro

doi: 10.1074/jbc.m113.462135pmid: 23658014

<p>Chitinase C from <i>Ralstonia</i> sp. A-471 (Ra-ChiC) has a catalytic domain sequence similar to goose-type (G-type) lysozymes and, unlike other chitinases, belongs to glycohydrolase (GH) family 23. Using NMR spectroscopy, however, Ra-ChiC was found to interact only with the chitin dimer but not with the peptidoglycan fragment. Here we report the crystal structures of wild-type, E141Q, and E162Q of the catalytic domain of Ra-ChiC with or without chitin oligosaccharides. Ra-ChiC has a substrate-binding site including a tunnel-shaped cavity, which determines the substrate specificity. Mutation analyses based on this structural information indicated that a highly conserved Glu-141 acts as a catalytic acid, and that Asp-226 located at the roof of the tunnel activates a water molecule as a catalytic base. The unique arrangement of the catalytic residues makes a clear contrast to the other GH23 members and also to inverting GH19 chitinases.</p><p>Background: Chitinase C from <i>Ralstonia</i> sp. A-471 (Ra-ChiC) is a chitinase that was first found in glycohydrolase family 23.</p><p>Results: The crystal structure of Ra-ChiC exhibited a tunnel-shaped conformation in its active site.</p><p>Conclusion: The tunnel-shaped conformation is essential for a unique arrangement of the catalytic residues and substrate specificity.</p><p>Significance: This is the first report on the tunnel-shaped binding site of an inverting chitinase.</p>